Molecular typing of Salmonella enterica serovar typhi isolates from various countries in Asia by a multiplex PCR assay on variable-number tandem repeats

Abstract

A multiplex PCR method incorporating primers flanking three variable-number tandem repeat (VNTR) loci (arbitrarily labeled TR1, TR2, and TR3) in the CT18 strain of Salmonella enterica serovar Typhi has been developed for molecular typing of S. enterica serovar Typhi clinical isolates from several Asian countries, including Singapore, Indonesia, India, Bangladesh, Malaysia, and Nepal. We have demonstrated that the multiplex PCR could be performed on crude cell lysates and that the VNTR banding profiles produced could be easily analyzed by visual inspection after conventional agarose gel electrophoresis. The assay was highly discriminative in identifying 49 distinct VNTR profiles among 59 individual isolates. A high level of VNTR profile heterogeneity was observed in isolates from within the same country and among countries. These VNTR profiles remained stable after the strains were passaged extensively under routine laboratory culture conditions. In contrast to the S. enterica serovar Typhi isolates, an absence of TR3 amplicons and a lack of length polymorphisms in TR1 and TR2 amplicons were observed for other S. enterica serovars, such as Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Paratyphi A, B, and C. DNA sequencing of the amplified VNTR regions substantiated these results, suggesting the high stability of the multiplex PCR assay. The multiplex-PCR-based VNTR profiling developed in this study provides a simple, rapid, reproducible, and high-resolution molecular tool for the epidemiological analysis of S. enterica serovar Typhi strains.

FIG. 1.

Gel electrophoresis of the TR1, TR2, and TR3 VNTR loci of 36 representative isolates amplified using the TR1, TR2, and TR3 primer pairs. Lanes: 1 to 14, Singapore isolates (lanes 8 and 12 are paired isolates collected 17 days apart); 15 to 22, Indonesian isolates; 23 to 27, Indian isolates; 28 to 32, Bangladeshi isolates (lanes 30 and 31 are paired isolates collected 38 days apart); 33 and 34, Nepalese isolates; 35 and 36, S. enterica serovar Typhi CT18 and TY2-b; M, 100-bp DNA marker.

FIG. 2.

Agarose gel analysis of the VNTR banding profiles amplified from the 36 representative S. enterica serovar Typhi isolates by multiplex PCR containing TR1, TR2, and TR3 primer pairs. Lanes: 1 to 14, Singapore isolates (lanes 8 and 12 are paired isolates collected 17 days apart); 15 to 22, Indonesian isolates; 23 to 27, Indian isolates; 28 to 32, Bangladeshi isolates (lanes 30 and 31 are paired isolates collected 38 days apart); 33 and 34, Nepalese isolates; 35 and 36, S. enterica serovar Typhi CT18 and TY2-b; M, 100-bp DNA marker.

FIG. 3.

Specificity of the multiplex PCR assay for strain typing S. enterica serovar Typhi isolates. Lanes: 1 to 9, various S. enterica serovar Typhi isolates; 10, S. enterica serovar Typhimurium TR7095; 11, S. enterica serovar Typhimurium SL1344; 12, S. enterica serovar Enteritidis LK5; 13, S. enterica serovar Enteritidis PT4; 14, S. enterica serovar Paratyphi A; 15, S. enterica serovar Paratyphi B; 16, S. enterica serovar Paratyphi C; M, 100-bp DNA marker.